Improved plant varieties have been obtained by ‘classical’ crossbreeding ever since man exchanged nomadic existence for permanent settlement. In the more recent history, scientists started to unravel the behavior of genetic material during crossings and plant breeders could, and still do benefit from the knowledge contained within the Mendelian laws predicting the distribution of a given genetic trait in the offspring of a crossing. With the advent of plant molecular biology plant breeders can, with an ever increasing precision, insert novel chimeric genes into the genome of a plant. A variety of techniques is nowadays available to mediate genetic transformation of plants including agrolistics, microinjection, electroporation, direct gene transfer and bombardment with DNA-coated particles. A preferred and widely used plant transformation system makes use of the soil bacterium Agrobacterium (Zupan and Zambryski 1995, Gelvin 1998a, Gheysen et al. 1998). Nowadays, Agrobacterium is not only used to transform plants but also to transform yeast, moulds and filamentous fungi (Bundock et al. 1995, de Groot et al. 1998, Gouka et al. 1999, WO98/45455). It has furthermore been shown that components of the T-DNA production and transfer machinery of Agrobacterium are useful for import of DNA into nuclei of mammalian cells, opening perspectives for use of these components in gene therapy (Ziemienowicz et al. 1999). Agrobacterium transfers into a eukaryotic cell nucleus any DNA located on the T-DNA. This T-DNA is part of the wild-type Ti- (in case of Agrobacterium tumefaciens) or Ri-plasmid (in case of A. rhizogenes). Wild-type T-DNA carries the genes causing, after integration in the plant genome, crown gall tumors or the hairy root syndrome in case of infection with A. tumefaciens or A. rhizogenes, respectively. Also located on the wild-type Ti- or Ri-plasmids are vir genes (virulence genes) which are activated by plant phenolic compounds. Products of the vir genes are responsible for the transfer of the T-DNA into the eukaryotic genome. For transformation purposes, the T-DNA is disarmed (i.e. all disease-causing genes are removed) and vir genes are supplied either in trans on a helper plasmid (the T-DNA encompassing heterologous gene(s) is then located on a second binary plant transformation vector) or in cis in case of a co-integrate plant transformation vector. The heterologous genes of interest are cloned in between the two T-DNA 22 bp (in case of octopine Ti plasmids) or 25 bp (in case of nopaline Ti plasmids) imperfect border core sequences constituting to the right border (RB) and the left border (LB), that are the only in cis elements necessary to direct T-DNA processing. The border core sequences in RB and LB are organized as imperfect repeats.
The VirD1 and VirD2 proteins produce a single-stranded nick between the third and fourth base in the bottom strand of each border repeat (Yanofsky et al. 1986). Increased levels of VirD1 and VirD2 enhance the production of T-DNA complexes inside Agrobacterium and result in an increased plant transformation efficiency (Wang et al. 1990).
For many years, it was believed that only the DNA between the repeats, the T-DNA, and not the vector DNA external to the T-DNA was transferred to the eukaryotic cell. However, recent and more detailed characterization of the DNA inserts in transgenic plants demonstrates that also vector backbone sequences integrate very frequently into the plant genome (Martineau et al. 1994, Ramanathan and Veluthambi 1995, Cluster et al. 1996, van der Graaff et al. 1996, Kononov et al. 1997, Wenck et al. 1997, Wolters et al. 1998).
The authors of the present invention have previously found that the frequency of integration of vector sequences is not influenced by the plant species or the transformation method used. This is consistent with the view that transfer of vector backbone sequences is the consequence of read-through past the LB, a process that is occurring within the Agrobacterium cells and is most probably determined by factors within these cells. It should, however, be noticed that others reported vector backbone integration in 33% of Arabidopsis transformants obtained via root transformation and in up to 62% of transformants obtained via vacuum infiltration (Wenck et al. 1997). This implies that the transformation method used could be another factor influencing the frequency of vector backbone integration. Integration of vector backbone sequences has been reported to occur in many plant species including Petunia (Virts and Gelvin 1985, Cluster et al. 1996), Arabidopsis (Van der Graaff et al. 1996, Wenck et al. 1997), tobacco (Ramanathan and Veluthambi 1995, Kononov et al. 1997, Wenck et al. 1997), and potato (Wolters et al. 1998). Vector backbone integration is apparently independent of the type of Agrobacterium strain used for plant transformation (Kononov et al. 1997).
The inventors have previously analyzed different series of transformants for the presence of vector backbone sequences by using specific PCR reactions and DNA gel blot analysis. Three different transformation methods in two different plant species were evaluated, namely Arabidopsis thaliana root and leaf transformation and Nicotiana tabacum protoplast and leaf transformation. Finally, the influence of the replicon type, the ColE1 and pVS1 replicons, was evaluated. The results showed that neither the plant species nor the explant type used for transformation, the replicon type or the selection have a major influence on the frequency with which integration of vector sequences occurred. In the past, it was postulated that this transfer of vector DNA that does not belong to the T-DNA could be the result of read-through at the left border, which would prevent the normal termination of the T-DNA transfer. Alternatively, DNA transfer could start at the left border and proceed towards the right border (Ramanathan and Veluthambi, 1995; van der Graaff et al., 1996). In the transgenic plants described above, however, it was observed that many contain vector backbone sequences linked to the left border as well as vector junctions with the right T-DNA border. DNA gel blots indicate that in most of these plants the complete vector sequence is integrated. Therefore, it was postulated that integration in the plant genome of complete vector backbone sequences can be the result of a conjugative transfer initiated at the right border and subsequent continued copying at the left and right borders, called read-through. This model implies that the left border is not frequently recognized as an initiation site for DNA transfer and that the right border is not efficiently recognized as a termination site for DNA transfer. These observations comply with the results of previous work showing that the right border region is intrinsically more active than the left border region in promoting T-DNA transformation (Jen and Chilton 1986a,b, Caplan et al. 1985). From all available data, it can be concluded that T-strand formation starts much more frequently at the right border than at the left border region.
In the future, it will be of utmost importance to prevent or to cure vector backbone integration as a consequence of Agrobacterium-mediated transformation. Firstly, regulatory authorities are demanding that transgenic plants to be released on the common marketplace are free of vector backbone sequences. Such backbone sequences can carry bacterial origins of replications, bacterial antibiotic resistance genes and possibly a number of other (foreign) genes. The same rigorous concerns will also be expressed by consumers who are becoming increasingly aware of such potential hazards associated with plant biotechnology. Secondly, also from a scientific point of view it is desirable not to have vector backbone integration in the genome of plants. Such sequences can influence transgene expression (Iglesias et al. 1997, Matzke and Matzke 1998, Jakowitsch et al. 1999). Vector backbone integration is also likely to interfere with T-DNA tagging experiments. Tags can be considerably longer than expected (Martineau et al. 1994) and vector backbone integration might also be the explanation for the fact that in a large percentage of T-DNA tagged Arabidopsis plants the T-DNA is not co-segregating with a mutant phenotype (Errampali et al. 1991, Feldmann 1991, Koncz et al. 1992, Van Lijsebettens et al. 1991).
Although the phenomenon of occasional vector backbone integration was already encountered as early as 1982 by Ooms et al., it lasted till 1995 for a first solution to be suggested by Ramanathan and Veluthambi (1995): “ . . . novel binary vectors with ‘stop-transfer’ signals adjacent to the left border may be constructed”. Since then, one has tried to understand the mechanism of vector backbone integration. A possible reason was discussed by Wenck et al. (1997): “ . . . inefficient nicking may be due to low amounts of virulence proteins, primarily VirD2”. Only very recently, however, methods were disclosed that prevent read-through at the T-DNA borders (WO99/01563) and by Hanson et al. (1999). These methods are based on including sequences outside the borders. These sequences are either genes coding for toxic compounds or sequences capable of interacting with DNA-binding proteins or sequences that are enriched in G+C nucleotides. A major drawback of the methods described in WO99/01563 and Hanson et al. (1999) is that the regeneration of plant transformants carrying more than the T-DNA region in their genome is prevented. It is conceivable that such methods impair the overall transformation efficiency, i.e. lower numbers of transformants will be obtained from a given transformation experiment. It was indeed reported by Hanson et al. (1999) that tobacco transformation efficiencies drop by as much as 30%. Nevertheless, Hanson et al. (1999) described their approach as a useful tool ‘for the elimination of non-T-DNA sequences from transgenic individuals’.
The current invention describes a solution to the technical problem of undesired vector backbone integration and provides advantages over existing methods.